mc3t3 e1 cells  (ATCC)

Journal: bioRxiv

Article Title: Label-free imaging of matrix mineralization in alginate-encapsulated bone spheroids using Coherent Raman Scattering microscopy

doi: 10.64898/2026.01.27.701971

Figure Lengend Snippet: Analysis of mineralized matrix deposition in MC3T3-E1 monolayer cell cultures (A) and MC3T3-E1 alginate-encapsulated bone spheroids (B) after 4 weeks of culture, using colorimetric and fluorescent assays. In monolayer cell cultures (A), bright field images show the presence of dark material deposited on top of the cell layer in OM-cultured cells. These deposits were positively stained for ARS, calcein and OsteoImage™, confirming that matrix mineralization was taking place. RM-cultured cells were negative for all staining methods. In spheroids (B), bright field images did not provide any information regarding the mineralization state of the sample. While OM-cultured spheroid were positively stained for ARS, fluorescent signal was detected in both RM- and OM-cultured spheroids supplemented with calcein, with a stronger signal observed in OM-cultured spheroids. OsteoImage™ stained samples did not provide any relevant information.

Article Snippet: MC3T3-E1 subclone 4 (ATCC, CRL-2593) were cultured in standard conditions at 37 ° C and 5% CO 2 in minimum essential alpha medium (MEM- α without ascorbic acid; ThermoFisher, A1049001) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich).

Techniques: Cell Culture, Staining

Journal: bioRxiv

Article Title: Label-free imaging of matrix mineralization in alginate-encapsulated bone spheroids using Coherent Raman Scattering microscopy

doi: 10.64898/2026.01.27.701971

Figure Lengend Snippet: SRS and epi-SHG images of mineral deposits and collagenous matrix deposited by MC3T3-E1 cells after 4 weeks of culture in OM. A) SRS image at a Raman shift of 960 cm −1 to detect the presence of mineral deposits by probing the phosphate groups. B) Epi-SHG image of the deposited collagen matrix (green). C) Overlay of the SRS and epi-SHG images to visualize the localization of the mineralized region in relation to the localization of the collagen matrix.

Article Snippet: MC3T3-E1 subclone 4 (ATCC, CRL-2593) were cultured in standard conditions at 37 ° C and 5% CO 2 in minimum essential alpha medium (MEM- α without ascorbic acid; ThermoFisher, A1049001) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich).

Techniques:

Journal: bioRxiv

Article Title: Label-free imaging of matrix mineralization in alginate-encapsulated bone spheroids using Coherent Raman Scattering microscopy

doi: 10.64898/2026.01.27.701971

Figure Lengend Snippet: TEM images of mineralized ECM in MC3T3-E1 monolayer cell cultures after 3 weeks of culture in OM. A) Low magnification image showing the cell (C), nuclei (N), and ECM organization, with collagen fibrils (*) and mineral deposits (arrow). B) High magnification image of an area of highly organized collagen fibrils (*), cells (C) and nuclei (N). C) High magnification image showing electron-dense mineral deposits (arrow) within the deposited collagen (*), indicating mineralization of the ECM.

Article Snippet: MC3T3-E1 subclone 4 (ATCC, CRL-2593) were cultured in standard conditions at 37 ° C and 5% CO 2 in minimum essential alpha medium (MEM- α without ascorbic acid; ThermoFisher, A1049001) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich).

Techniques:

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Osteoblasts respond to PTH treatment with increased Slit3 transcription and translation. mRNA expression of the Slit3 in the endplate tissue of WT young mice treated with Veh and aged mice treated with Veh or PTH for 1 month ( a ), and WT LSI mice treated with veh or PTH for 2 months ( b ) ( n = 3, one-way ANOVA with Tukey’s multiple comparisons test). c mRNA expression of the Slit3 in MC3T3 cells cultured in osteoblast-stimulated medium and subsequently treated with Veh or PTH at different doses for 3 days ( n = 3, t -test). d , e Protein expression level of Slit3 in MC3T3 cells cultured in osteoblast-stimulated medium and treated with Veh (PBS) or PTH for 3 days. ( n = 3, t -test). Representative images ( f ) and quantification ( g ) of IF staining of PGP9.5 positive primary DRG neuron fibers crossing the microgroove barrier (microfluid assay) cultured in condition medium (CM), CM + PTH, CM + PTH plus mouse Slit3 antibody (1 µg/mL), or CM + Veh (1X PBS) plus human recombinant Slit3 (1.25 µg/mL) for 1 week. Representative images showing co-immunostaining for OCN (green) and Slit3 (red) in the lumbar spine sections and quantitative analysis of number of Slit3 + cells in trabecular bone (TB) and endplate (EP) of aged mice ( h – j ) and PPR OCN −/− LSI mice ( k – m ) treated with PTH or Veh for 2 months. Scale bar: 100 µm. ( n ≥ 5, t -test). * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001

Article Snippet: MC3T3 subclone 4 cell line was purchased from ATCC (CRL-2593 TM ) and cultured using Alpha Minimum Essential Medium with ribonucleosides, deoxyribonucleosides, 2 mmol/L L-glutamine and 1 mmol/L sodium pyruvate, but without ascorbic acid (A10490-01, Thermo Fisher).

Techniques: Expressing, Cell Culture, Staining, Recombinant, Immunostaining

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Transcriptional mechanism underpinning Slit3 secretion. a mRNA expression levels of Ets1, E47, FoxJ2 , and FoxA2 genes in MC3T3 cells cultured in osteoblast differentiation-inducing medium and treated with vehicle or PTH (100 nmol/L) for 3 days ( n = 3, t -test). b , c Protein expression level of E47, FoxA2 in MC3T3 cells cultured in stimulated medium and treated with PTH or Veh (PBS) for 3 days ( n = 3, t -test). Representative images depicting E47 IF staining (green) in lumbar vertebral body and endplate sections ( d ), quantitative analysis of the number of E47 + cells in the vertebral body ( e ) and endplate ( f ) of aged mice treated with Veh or PTH ( n = 5, t -tset). Scale bar: 100 µm. Representative images showing FoxA2 IF staining (red) in lumbar vertebral body and endplate sections ( g ), quantitative analysis of the number of FoxA2 + cells in the vertebral body ( h ) and endplate ( i ) of aged mice treated with PTH or vehicle for 2 months ( n = 5, t -tset). Scale bar: 100 µm. j Relative fold enrichment of the E47 binding site in the Slit3 promoter region from stimulated MC3T3 cells treated with vehicle or PTH (100 nmol/L) for 3 days ( n = 3, two-way ANOVA with Sidak’s multiple comparisons test). k Relative fold enrichment of the FoxA2 binding site in the Slit3 promoter region from stimulated MC3T3 cells treated with vehicle or PTH (100 nmol/L) for 3 days ( n = 3, two-way ANOVA with Sidak’s multiple comparisons test). * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001

Article Snippet: MC3T3 subclone 4 cell line was purchased from ATCC (CRL-2593 TM ) and cultured using Alpha Minimum Essential Medium with ribonucleosides, deoxyribonucleosides, 2 mmol/L L-glutamine and 1 mmol/L sodium pyruvate, but without ascorbic acid (A10490-01, Thermo Fisher).

Techniques: Expressing, Cell Culture, Staining, Binding Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Effects and mechanisms of iron overload on the proliferation and differentiation of preosteoblastic cells via a 3D microsphere culture system

doi: 10.3389/fbioe.2026.1700858

Figure Lengend Snippet: The 3D microsphere culture system attenuates proliferation and ROS expressions in preosteoblastic cells under iron overload. (A) Live/dead staining of cells cutured on different groups. (B) Intracellular ROS staining of preosteoblastic cells cultured on different groups. (C) The corresponding quantitative analysis of live/dead fluorescence. (D) The corresponding quantitative analysis of fluorescence intensities. (E) Growth curve of preosteoblastic cells cultured on TCP and GM groups with α-MEM without FAC. (F) The corresponding quantitative analysis of ratio of early to late apoptosis. (G) Apoptosis analysis of preosteoblastic cells cultured on different groups via Annexin V-FITC/PI flow cytometry. (H) Cell viability of preosteoblastic cells exposure to 50/300 μM FAC for 24/36/72 h. (I) Cell cycle distribution of preosteoblastic cells cultured on different groups via flow cytometry. (J) The corresponding quantitative analysis of cell cycle phase proportions (G1/G2/S). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Mouse preosteoblastic cells (MC3T3-E1 subclone 4; ATCC) were cultured in α-minimum essential medium (α-MEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: Staining, Cell Culture, Fluorescence, Flow Cytometry

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Effects and mechanisms of iron overload on the proliferation and differentiation of preosteoblastic cells via a 3D microsphere culture system

doi: 10.3389/fbioe.2026.1700858

Figure Lengend Snippet: The 3D microsphere culture system attenuates integrin-mediated mechanotransduction in preosteoblastic cells under iron overload. The immunofluorescence staining of (A) ITGA1 and (B) ITGB1 of cells cultured on different groups. And the corresponding quantitative analysis of the mean fluorescence intensity (MFI) for (C) ITGA1, (D) ITGB1 and (E) F-actin. (F) The mechanotransduction-related gene expressions of cells cultured on different groups. (G) The corresponding quantitative analysis of the absolute reduction of mRNA expression for mechanotransduction-related genes. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Mouse preosteoblastic cells (MC3T3-E1 subclone 4; ATCC) were cultured in α-minimum essential medium (α-MEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: Immunofluorescence, Staining, Cell Culture, Fluorescence, Expressing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Effects and mechanisms of iron overload on the proliferation and differentiation of preosteoblastic cells via a 3D microsphere culture system

doi: 10.3389/fbioe.2026.1700858

Figure Lengend Snippet: The 3D microsphere culture system attenuates osteogenic function impairment in preosteoblastic cells under iron overload. The immunofluorescence staining of (A) OPN and (B) OCN of cells cultured on different groups. And the corresponding quantitative analysis of the MFI for (C) OPN, (D) OCN. (E) Alizarin Red (calcium nodule formation), alkaline phosphatase (ALP, early osteogenic marker), and Sirius Red (collagen deposition) staining. Quantification of (F) Alizarin Red staining, (G) ALP staining, (H) Sirius Red. (J–I) The osteogenic - related gene expressions of cells cultured on different groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Mouse preosteoblastic cells (MC3T3-E1 subclone 4; ATCC) were cultured in α-minimum essential medium (α-MEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: Immunofluorescence, Staining, Cell Culture, Marker

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Effects and mechanisms of iron overload on the proliferation and differentiation of preosteoblastic cells via a 3D microsphere culture system

doi: 10.3389/fbioe.2026.1700858

Figure Lengend Snippet: RNA sequencing analysis of preosteoblastic cells cultured on GM + FAC and TCP + FAC. (A) Volcano plot of DEGs between GM and TCP groups. (B,C) KEGG pathway enrichment analysis of upregulated and downregulated genes. (D) Cluster heatmap of DEGs in GM and TCP, with low expression indicated in blue and high expression in orange. (E,F) GO enrichment analysis of upregulated and downregulated genes between the two groups. (G–L) GSEA enrichment analysis of inter-group signaling pathways.

Article Snippet: Mouse preosteoblastic cells (MC3T3-E1 subclone 4; ATCC) were cultured in α-minimum essential medium (α-MEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: RNA Sequencing, Cell Culture, Expressing, Protein-Protein interactions

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Effects and mechanisms of iron overload on the proliferation and differentiation of preosteoblastic cells via a 3D microsphere culture system

doi: 10.3389/fbioe.2026.1700858

Figure Lengend Snippet: RNA sequencing analysis of preosteoblastic cells cultured in the same culture system (TCP vs TCP + FAC, GM vs GM + FAC). (A,B) GO enrichment analysis for upregulated and downregulated genes of TCP + FAC and TCP. (C,D) KEGG pathway enrichment analysis for upregulated and downregulated genes of TCP + FAC and TCP. (E,F) GO enrichment analysis for upregulated and downregulated genes of GM + FAC and GM. (G,H) KEGG pathway enrichment analysis for upregulated and downregulated genes of GM + FAC and GM.

Article Snippet: Mouse preosteoblastic cells (MC3T3-E1 subclone 4; ATCC) were cultured in α-minimum essential medium (α-MEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: RNA Sequencing, Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Effects and mechanisms of iron overload on the proliferation and differentiation of preosteoblastic cells via a 3D microsphere culture system

doi: 10.3389/fbioe.2026.1700858

Figure Lengend Snippet: The schematic diagram of integrin mediated-signal pathway regulations on preosteoblastic cells under iron overload in 3D cell culture.

Article Snippet: Mouse preosteoblastic cells (MC3T3-E1 subclone 4; ATCC) were cultured in α-minimum essential medium (α-MEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: Cell Culture